Antibodies, and methods for their use

ABSTRACT

This invention relates to antibodies and is particularly, though not exclusively, concerned with diagnostic and therapeutic methods using monoclonal bi- or tri-specific antibodies. The invention also provides a method in which binding of a first antigen to a first antibody binding site causes release of a second antigen from an adjacent second antibody antigen binding site.

This invention relates to antibodies and is particularly, though notexclusively, concerned with diagnostic and therapeutic methods usingmonoclonal or polyspecific, such as bi-or tri-specific antibodies.

Monoclonal-based antibody assays have not achieved full potential asthey generally have to be performed by trained operators in alaboratory. Even relatively simple assays require washing steps andmultiple manual addition of reagents. There is a need for a one-stepsystem, which would have a wide field of applications.

Monoclonal antibodies have also found application in treatment ofdisease. For example, monoclonal antibody conjugates have been used tolocalize and treat tumours in the body, destroying the tumour with toxicagents, including ricin and radioiodine, attached to the antibodyprotein.

Bispecific antibodies have been developed from monoclonal antibodytechnology, in the example of bispecific immunoglobulin G eachbispecific antibody has two antigen binding sites of differingspecificities. Bispecific antibodies can be produced by fusing twodifferent hybridomas which respectively secrete monoclonal antibodiesagainst the antigens of interest to form a single hybrid-hybridoma or"fusoma", (sometimes called a "polydoma") (Songsivilai, S and LachmannP.J (1990) Clin Exp Immunol 79, 315 and Suresh MR et al (1986) Proc.Natl Acad Sci USA 83, 7989 and GB2169921A). Parent hybridomas can beremoved by standard HAT selection or introduction of selectable drugresistance (De Lau B.M, et al (1989) J. Immunol Methods 117 ,1). Thefirst bispecific antibodies produced were used in a conventionalimmunoassay (Milstein C., and Cuello A.C., (1983) Nature 305 537). Theantibodies were produced by fusing a monoclonal antibody-secreting cellwith splenocytes from an immune mouse. The first binding site wasspecific for an antigen of interest. The second binding site of theenzyme was specific for a marker enzyme. The immunoassay demonstratedincreased assay sensitivity, reduction in signal-to-noise ratio,simplification of staining procedures, and preservation ofultrastructural detail.

Bispecific antibodies have found extensive application in novel therapyregimes targeting effector toxins, which remain bound to the antibody,to rumours (Corvalan JRF et al (1987) Cancer Immunol Immunother 24 133),crosslinking cellular antigens on cytotoxic killer cells to rumourtargets (Nitta T., et al (1990) Lancet 335 368) and Fanger MW and GuyrePM, Tibtech 9, 375-380 (1991). Other methods of producing bi-andtrispecific antibodies, such as chemical linkage, are reviewed in thelatter paper.

WO90/07714 discloses an immunoassay in which an enzyme is stabilised bybinding to a bispecific antibody against heat denaturation. W091/09134discloses a bispecific antibody capable of binding both to an enzymethat converts an inactive anticancer prodrug into its active form and toa human cancer cell. An immunocomplex comprising the antibody and theenzyme can be administered to cancer patients together with the inactiveprodrug to selectively kill cancer cells with minimal side effects. Theenzyme remains bound to the antibody in an active form. Also disclosedare methods of producing polydomas.

It is an object of the invention to provide an immunoassay methodinvolving fewer, preferably only one, reaction steps than conventionalimmunoassay methods.

According to one aspect of the invention, there is provided a method inwhich binding of an antigen to one antibody antigen binding site causesrelease of another antigen from an adjacent second antibody antigenbinding site. Whilst not wishing to be bound by theory the applicantsbelieve that steric hindrance between the incoming antigen and the boundantigen causes release of the bound antigen from the second antibodybinding site. The first and second antibody antigen binding sites may beprovided by the same multispecific antibody or by different antibodieswhich are physically adjacent. The term "multispecific" embraces allantibodies having more than one antigen binding site such as bispecificand trispecific antibodies. The release of a bound molecule from thesecond site through binding of another molecule to a first site, may betermed antibody-mediated signal transduction. The term "antibody" usedherein embraces immunoglobulins such as IgG, IgA, IgM, IgD and IgE andother proteins having the antigen-binding properties of anaturally-occurring antibody or antibodies produced by recombinant DNAtechnology or any other such methods.

We have surprisingly found that where antibodies are coated on amicrotitre tray at very high concentrations, for example greater than10-100 μgml⁻¹, as compared to standard concentrations which aretypically 1-5 μgml⁻¹, such that the antibodies are arranged in veryclose proximity to each other binding of an antigen to one antibodyantigen binding site can cause release of another antigen bound to anadjacent second antibody antigen binding site. In a preferred embodimentan immunoassay comprises binding antibody to a surface at aconcentration of protein of greater than about 20 μgml⁻¹ preferablygreater than about 50 μg ml⁻¹.

The second antigen may be bound in an inactive form by the secondantibody antigen binding site and released in an active form on bindingof the first antigen to the first antibody antigen binding site. Thesecond antigen may be a drug or other therapeutic agent, or an enzyme.The enzyme may be for example β-galactosidase, or urease.

Monoclonal antibodies have been reported which block the action ofcancer therapy drugs. For example antibody NO-1 neutralizes thecytotoxic action of mitozantrone, a potent anti-cancer drug. (FlayellSU, Flavell DJ(1991) Br.J.Haematol 78, 330-3). A bispecific antibody inaccordance with the invention, with one site directed against a drug,inactivating that drug, will release active drug at the site ofexpression of the molecule to which a second antigenic site of thebispecific antibody is directed.

Release of the antigen from the second antigen binding site may lead tobinding of the released antigen, or one of its reaction products if itis, for example, an enzyme or other catalytic molecule, or a reactionproduct of a reaction catalysed by it, at a third site on an adjacentantibody causing release of a bound third antigen from an adjacentfourth antigen binding site.

A diagnostic multispecific antibody may induce release of a therapeuticagent in a second "therapeutic" type multispecific antibody by cascadeaction where the binding of a diagnostic indicator to a first bindingsite of the first antibody results in release of an enzyme already boundto the first enzyme at a second binding site, the released enzyme or oneof its reaction products binding to a second antibody, and thissecondary binding event then causes release of a therapeutic agent boundby the second antibody. Binding of a reaction product of the enzyme ispreferred as this will produce amplification of the initial bindingsignal. In a trispecific antibody for diagnostic/therapeutic use whichalso operates in a cascade action, the first antigen binding site may bedirected to the diagnostic marker, the second against an indicatorenzyme and the third antigen binding site carrying a therapeutic agentin an inactive form. It will be appreciated that the antibody can betailored to suit the application. For example an IcM antibody may beused which features ten different reaction steps.

In a preferred embodiment one antigen binding site holds the diagnosticor therapeutic agent in an inactive form by molecular binding at or nearthe site of indicator/therapeutic activity, such as the active site of acatalytic enzyme or the molecular component essential for therapeuticdrug action. In a diagnostic method the second antigen binding site isdirected against the molecule under test, be it a marker indicative of adisease or microorganism etc. In the presence of this marker, the agentheld in inactive form is released in an active form, resulting fromsteric hindrance from the close proximity of the two different antibodyantigen binding sites. In a therapeutic application the bound inactiveagent may be released by presence of the diagnostic molecule or otherantigen under test or a molecule or other antigen carried on abacterium, virus or other micro-organism against which treatment isbeing performed.

Further diagnostic uses of antibodies of the invention include:

Measurement of SP-A in amniotic fluid, pharyngeal aspirate, gutaspirate, blood, tissue section.

Assessment of risk of Respiratory Distress Syndrome, RDS, where absentor low levels of SP-A indicate risk.

Monitoring for appearance of lung function in infants suffering fromRDS, adults with adult RDS. Increasing levels of SP-A indicate normallung function.

Monitoring success of treatment of RDS with artificial surfactantreplacement therapy. Return of appearance of lung function ischaracterised by appearance of SP-A.

The release of two different bound molecules from different antibodiesmay give a reaction that only occurs when both released molecules arepresent. A reaction product may bind to a further antibody triggeringthe release of the substrate, e.g a therapeutic or diagnostic moleculebound to the further antibody. In a therapeutic application, twoprodrugs may be released which only become active for treatment whenboth are present, giving the active drug form. For example, in thetreatment of lung cancer a first bispecific antibody has a first antigenbinding site directed against lung surfactant apoprotein A ("SP-A")which is expressed by most lung tumours and a second antigen bindingsite directed against a prodrug A. A second bispecific antibody has afirst antigen binding site directed against a transferrin receptorindicative or rapid malignant growth and a second prodrug, prodrug B, inwhich the combined prodrugs A and B produce an active anticancercomplex. In use treatment of a lung cancer patient with a cocktailcomprising the two antibodies having bound prodrugs A and B will resultin release of the prodrugs A and B in the presence of SP-A and thetransferrin receptor to form the active anticancer complex. Indiagnostic applications, the presence of two different diagnosticantigens can trigger a cascade enzyme reaction, the detectablediagnostic indicator product only being produced when both diagnosticepitopes are detected. For example, two bispecific antibodies may beused having first antigen binding sites specific for inhibin A and Bchains respectively and second antigen binding sites specific for horseradish peroxidase and glucose oxidase respectively. In the presence ofboth inhibin A and B chains glucose is converted by glucose oxidaseproducing peroxide which is then converted by the peroxidase withreadily detectable substrate conversion of orthophenyldiamine. Inparallel with. conventional antigen capture assays in which twodifferent antigenic sites must be detected before a positive result isobtained.

The first and second antibody antigen binding sites may be provided by(i) a mixture of two monoclonal antibodies both at high concentrations,in excess of 20 μg ml⁻¹, preferably, 50 μgml⁻¹, for coating a surface inan immunoassay, (ii) fusoma, secreting parental monoclonal antibodies inaddition to a bispecific antibody and (iii) a bispecific antibody,having binding sites for different antigens, purified to homogeneity orproduced by chemical modification. Event (i) may be considered asintermolecular transduction, whilst (iii) may be considered asintramolecular transduction. Event (ii) involves inter- andintra-molecular transduction.

Diagnostic intermolecular signalling can be achieved economically by amixture of two preexisting monoclonal antibodies or by standardprocessing of unpurified bispecific antibody, for example, by affinitychromatography using Protein A or Protein G or ion exchange or gelfiltration to isolate secreted immunoglobulin.

Intramolecular antibody signalling requires bi-and tri-specificimmunoglobulin purified to homogeneity. Purification to homogeneity maybe achieved by sequential affinity chromatography steps, using anaffinity matrix against which each antigenic site is directed.

Thus, the multispecific antibody is purified by chromatography or ionexchange using immobilized enzyme, therapeutic drug or diagnosticmolecule.

Cost efficient affinity chromatography may be achieved by usingimmobilized anti-idiotypic antibody matrixes, where the immobilizedantibodies recognize an idiotypic determinant of the multispecificantibody undergoing purification to homogeneity.

According to another aspect of the invention there is provided animmunoassay method for determining the presence or absence of an antigenin a sample, the method comprising contacting the sample with amultispecific antibody having binding sites for the antigen and anenzyme, binding of the enzyme to the antibody inactivating the enzyme,inwhich binding of the antigen to the antibody results in release of boundenzyme from the antibody in an active form from the antibody anddetecting the activity of the released active enzyme which indicates thepresence of the antigen in the sample. Thus this aspect of the inventionprovides a simple immuneassay method involving a single reaction step.

Whilst not wishing to be bound by theory the applicants believe thatsteric hindrance between the incoming antigen and the bound enzymecauses release of the enzyme from the antibody. Therefore the enzyme istypically chosen on the basis of its size to facilitate steric hindrancewith the antigen of interest. The antibody used should bind the enzymein a sufficiently stable manner to ensure that the enzyme does notbecome unbound in the absence of the antigen. Preferably the enzyme isbound to the antibody by its active site.

The antigen may be for example SP- A, a lack of which is indicative of arisk of Respiratory Distress Syndrome occurring in the preterm prematureinfant (Hallman et al (1988)Am J Obs Gynecol 158,153). This respiratorycondition affects 2% of all newborn babies and is the most common causeof death in normally-formed babies in the first week of life.

The enzyme may be for example βgalactosidase, glucose oxidase, urease,carbonic anhydrase, or horseradish peroxidase, all of which are wellcharacterised and easily assayable enzymes.

According to another aspect of the invention there is provided amultispecific antibody having binding sites for an antigen and an enzymein which the enzyme is inactivated by binding to the antibody and isreleased from the antibody in an active form through binding of theantigen to the antibody. Preferably, the antibody is bispecific.

According to another aspect of the invention there is provided a methodof detecting SP-A in a sample of mammalian body fluid comprisingcontacting the sample with a multispecific antibody having binding sitesfor SP-A and an enzyme, binding of the enzyme to the antibodyinactivating the enzyme, in which binding of SP-A to the antibodyresults in release of enzyme in an active form from the antibody anddetecting the presence of the released active enzyme which indicates thepresence of SP-A in the sample. The enzyme may be β-galactosidase.

According to another aspect of the invention there is provided animmunoassay method for determining the presence or absence of an antigenin a sample, the method comprising contacting the sample with a firstbispecific antibody having binding sites for the antigen and a firstenzyme, a reaction product of the first enzyme acting as a substrate fora second enzyme at a second site which catalyses a readily-detectablereaction indicating the presence of the antigen in the sample. The firstenzyme may be glucose oxidase. The second enzyme may be horseradishperoxidase.

Any diagnostic method in accordance with the invention may be arrangedto be carried out in a biosensor in which the multispecific antibodyacts as the biological sensing element of the biosensor. Hithertomonoclonal antibodies have been used in electrode biosensors to detecthuman gonadotrophin (Robinson G.A et al (1987) Biosensors 3, 147) andStaphylococcus aureus in food (Mirhabibollahi B, et al (1990) J. Appl.Bacteriol 68,577). General application has, however, proved impossibleas detector antibodies must be removed by washing before measurement ofantibody-bound antigen and also problems exist with enzyme regeneration.As the methods of the invention use an integral enzyme the bispecificantibody can be incorporated directly into electrodes and semiconductortransducers. For example an oxygen electrode or an ion selective fieldeffect transistor (ISFET) may include a bispecific antibody to which isbound glucose oxidase; or a urea electrode, or a chemically sensitivefield effect transistor (CHEMFET) may include a bispecific antibody towhich is bound urease.

Examples of enzymes and the preparation of multispecific antibodieswhich may be used in the method of the invention are now described belowby way of example only. β-galactosidase is well characterised and itsactivity can be easily assayed.

Glucose oxidase is isolated at low cost from Aspergillus niger and has amolecular weight of 186kD. Glucose oxidase is a mannose-richglycoprotein and consequently can be cross-linked to increase the localconcentration of bound inactive enzyme through the mannose carbohydratechain with retention of enzyme activity. (Kozulic B. et al (1987) ApplBiochem Biotechnol 15 265). The size of glucose oxidase polymers can becontrolled by the chemical reaction. Glucose oxidase can be used as theenzyme component of an oxygen electrode.

Urease, which can be isolated from Jack beans at low cost, is ahexameric protein of 590kD, with one active site in each 96kD subunit.Urease is used as the enzyme component in the urea electrode.

Carbonic anhydrase is a monomeric enzyme with a relatively low molecularweight of 29kD. Carbonic anhydrase catalyses carbon dioxide hydrationand hydrogen carbonate dehydration and can be isolated from human redblood cells at low cost.

Horseradish peroxidase has a well characterised heme site (La Mar GN etal (1980)J Biol Chem 255, 6646). Horseradish peroxidase may be used in atwo site immunoassay method with glucose oxidase at a first site andhorseradish peroxidase at a second site to produce an enzyme cascadewith the hydrogen peroxide produced by glucose oxidase acting as asubstrate for horseradish peroxidase.

The preparation of antibodies in accordance with the invention andmethods of their use will now be described, by way of example only withreference to the accompanying FIGS. 1 to 5 in which:

FIG. 1 illustrates the operation of an antibody in accordance with theinvention;

FIG. 2 illustrates applications of antibodies in accordance with theinvention;

FIG. 2A shows a diagnostic embodiment employing a bispecific antibodywhich binds analyte and enzyme. FIG. 2B shows a therapeutic embodimentemploying a bispecific antibody which binds a cancer cell surfaceantigen and an anti-cancer drug. FIG. 2C shows a combineddiagnostic/therapeutic application employing two bispecific antibodies.One antibody binds both a cancer cell antigen and an enzyme. The otherantibody binds the enzyme or a reaction product thereof, as well as aanti-cancer drug.

FIG. 3 is a graph illustrating the activity of enzyme released from anantibody in accordance with the invention;and

FIG. 4 is a graph illustrating the activity of enzyme released from anantibody in accordance with the invention; and

FIG. 5 is a graph illustrating the activity of enzyme released from anantibody in accordance with the invention.

The bispecific antibody 10 of immunoglobulin G type shown in FIG. 1comprises first and second binding sites 12 and 14 which bind in usefirst and second antigens 16 and 18 respectively. Binding of the firstantigen 16 to first antigen binding site 12 causes release of boundsecond antigen 18 from the second binding site 14.

In the diagnostic application shown in FIG. 2A) bispecific antibody 20has first and second antigen binding sites 22,24 directed respectivelyto an analyte of interest 26, e.g. SPA and to an enzyme 28 which hasreadily detectable substrate conversion activity e.g. β-galactosidase.The enzyme is inactivated when bound to the antibody at the secondbinding site 24 for example by binding through or adjacent its activesite or through alteration of the active site's configuration.

Binding of the analyte 26 from a sample to the first binding site 22causes release of the bound enzyme 28 into the media where it's activitycan be readily detected indicating presence of the analyte.

In the therapeutic application illustrated in FIG. 2B) bispecificantibody 30 has first and second antigen binding sites 32,32 directedrespectively to an antigen on the surface of a cancer cell 36, and to ananti-cancer drug 38 . The drug 38 is inactivated when bound to theantibody at the second binding site 34. Binding of the antigen 36 to thefirst binding site 32 causes release of the bound drug 38 in an activeform whereupon it can act against the cancer cell expressing antigen 36.

In the combined diagnostic/therapeutic application shown in FIG. 2C) twodifferent bispecific antibodies 40,42 are used.

Antibody 40 has specificity for an antigen 44 carried by a cancer celland for an enzyme 46 which it binds in an inactive form. Antibody 42 hasspecificity for an anticancer drug 48 and for the enzyme 46 or areaction product of the enzyme. Binding of the antigen 44 to antibody 40causes release of the enzyme 46 in an active form. The activity of thereleased enzyme can be readily detected. The enzyme 46 or one of itsreaction products then binds to the second antibody 42 which causesrelease of the drug 48 in an active form to kill the cancer cellexpressing antigen 44.

Bispecific antibodies are conveniently prepared by hybridoma cell fusiontechnology. First suitable monoclonal antibody secreting hybridoma cellsare isolated and characterised. Next the parental cell lines arerendered drug resistant by growth in various selection media. These drugresistant clones can then be used for bispecific antibody production bycell fusion between parental cell lines of differing drug resistant orbetween a drug resistant hybridoma and splenocytes from an immune mouse.After cell fusion and selection, cultures are screened for production ofantibodies of the desired reactivities. Chosen cultures are cloned andsecretion of bispecific immunoglobulin by the fusoma confirmed byimmunoassay.

For use in the current invention, secreted immunoglobulin is enriched byprotein A affinity chromatography. The enriched antibody is thensubjected to sequential affinity chromatography steps to isolatehomogeneous bispecific immunoglobulin.

Preparation of Bispecific Antibodies

A) Preparation of hybridomas secreting suitable monoclonal antibodies

Bispecific antibodies are conveniently prepared by fusoma technology.

First, monoclonal antibody-secreting cell lines are isolated against theenzymes of interest and against the cytotoxic drug methotrexate.

Methotrexate is coupled to ovalbumin for increased immunogenicity onantigen (enzyme) presentation. For immunisation, enzymes are used innative form and in conyugates with keyhole limpet haemocyanin, forenhanced immunogeneicity. Serum responses of immunised BALB/C mice aremonitored and on generation of a suitable response hybridomas areprepared by cell fusion of splenocytes from immune mice to mouse SP2/0myeloma cells. Hybridomas are screened initially against the targetantigen or methotrexate conjugate by enzyme-linked immunosorbent assay(ELISA). Monoclonal antibodies produced by cloned hybridomas secretingthe antibodies against the enzymes are then screened for ability toblock enzyme-mediated substrate conversion reactions. Monoclonalantibodies having the ability to block such reactions may do so throughbinding to or near the active site. Methotrexate-reactive antibodies arescreened for their ability to block the cytotoxic effect ofmethotrexate.

Hybridomas producing suitable monoclonal antibodies are then cultured intoxic media to isolate drug resistant clones suitable for fusomaproduction.

Two selectable markers are employed to develop suitable drug resistantclones for fusoma production. Hybridomas are cultured in 5μg per ml. of6-thioguanine, to select hypoxanthine guanosine phosphoribosyltransferase deficient variants.

To induce thioguanine resistance 4×10⁷ hybridoma cells were dispersedinto 6×48 well tissue culture plates containing alpha MEM medium(Stanners CP,Eliceri G and Green H 1971,Nature, New Biol 230,52) with10%(V/V) heat inactivated foetal calf serum (FCS),20% (V/V) conditionedmedium from J774 macrophage cell line (Cancer Research 1977 37,546) and5 μg per ml of 6-thioguanine (Sigma A4660). After approximately 3 weeks,clonal outgrowths of drug resistant clones were visible. Clones wereremoved by pipette and subcultured. Antibody secretion was confirmed andselected cultures stored. These variants are then selected against inthe standard HAT selection system (Littlefield J, W, (1964) Science 145,709).

Drug resistant cells are also selected by culture in increasingconcentrations of the cardiac glycoside ouabain which inhibits thesodium potassium ATPase of the mammalian plasma membrane. Wild typecells die in the presence of ouabain whilst resistant clones can grow in180-fold excess concentration of the drug (Mankovitz R et al (1974) Cell3, 221).

To induce ouabain resistance, 2×10⁶ hybridomas were grown andsubcultured at confluence in alpha MEM, 10%(v/v) FCS in increasingconcentrations of ouabain (Sigma 03125) from 1 μM to 0.5mM.

To induce double drug resistance(ie to ouabain and thioguanine)cellswere grown in increasing concentrations of ouabain as above. Oncecapable of growth in 0.5mM ouabain medium,drug resistance to6-thioguanine was induced as described above.

Hybridomas resistant to 6-thioguanine and to ouabain are cloned readyfor fusoma production.

b) Fusoma production

Fusomas secreting bispecific antibodies are produced by conventionaltechniques in a series of cell fusion experiments to select thoseproducing bispecific antibodies with an enzyme-reactive arm and a secondantibody binding site recognising the antigen of interest. The fusomasare derived from "enzxyme-reactive cells", whether splenocytes fromimmune mice or hybridomas, and from "antigen reactive cells". Examplesof antigen reactive cells include those producing the antibodies A15,recognising ovalbumin of 43kD, KLH1, recognising keyhole limpethaemocyanin of 800 kD and D4 and E8 both of which react with human lungsurfactant apeprotein A (SP-A) (Randle BJ et al (1992) in preparation).Antibody E8 is thought to be similar to antibody PE10 described inKuroki Yet al Am. J Pathol 1986 124; 25-33. Cell fusion experiments areperformed in three series:

1. Fusion of thioguanine resistant, HAT sensitive hybridomas antigenreactive or enzyme reactive to splenocytes of immune miceenzyme-reactive or antigen reactive selection by HAT.

2. Fusion of thioguanine resistant hybridomas either antigen orenzyme-reactive to ouabain resistant hybridomas, either antigen orenzyme-reactive selection by ouabain thioguanine medium.

3. Fusion of double resistant thioguanine/ouabain hybridomas eitherantigen or enzyme reactive to wild type hybridomas either antigen orenzyme reactive with selection in HAT ouabain medium.

Cell fusions are performed by standard techniques. Thioguanine-resistanthybridomas are mixed with splenocytes from immune mice in the ratio 1:10cells respectively (series 1) and fusoma cells prepared by incubation in50% (w/v) polyethylene glycol 1500 in serum free medium for 75 seconds.The cell fusion event is terminated by timed addition of serumcontaining growth medium. The fusomas are then plated out in multiwellplates, up to 800 separate cultures, and grown in HAT selection mediumfor two weeks.

Where fusomas are produced by fusion of two preexisting hybridomas withdiffering selectable markers (series 2) the cells are mixed in a 1:1ratio prior to fusion. The fusion event is performed in 50% (w/v)polyethylene glycol 1500 in serum free medium for 75 seconds. Thereaction is terminated by timed addition of serum containing medium over5 minutes. Fusomas are plated out in 200 separate cultures in multiwellplates and in selection medium containing 5 μg per ml thioguanine and0.5mM ouabain. Cultures are inspected for growth after two weeks inincubation at 37° C., 5% CO₂ (v/v) in air.

Where fusomas are produced by fusion of double drug resistant hybridomasto wild type hybridomas (series 3) the cells are mixed in a 1:1 ratioprior to fusion. The fusion event is performed in 50% (W/V) polyethyleneglycol 1500 in serum free medium for 75 seconds. The reaction isterminated by timed addition of serum containing medium over fiveminutes. Fusomas are plated out in 200 separate cultures in multiwellplates and in HAT selection medium containing 0.5 mM ouabain.

Cultures are then screened for recognition of the enzyme ormethotrexate. Reactive cultures are then tested for recognition of thechosen antigen. Cultures are screened by enzyme-linked immunosorbentassay (ELISA) for secretion of antibody reactive with the antigen ofchoice. Antigen is immobilised on 96-well multiwell plates at aconcentration of 5-10 μgml⁻¹ by incubation overnight at 4° C. in 0.1Mcarbonate buffer pH 9.6, 50 μl per well. Plates are blocked with 100 μlper well 10% (v/v) fetal calf serum in phosphate buffered saline (PBS)for 2 hrs at room temperature. Culture supernatants under test areloaded in duplicate at 50 μl per well and incubated for 1 hr at roomtemperature. The plates are washed with 0.05% (v/v) Tween 20 in PBS andbound antibody is detected using a second layer enzyme-conjugatedantimouse immunoglobulin antibody with subsequent detection for enzymesubstrate conversion. Cultures identified as secreting bispecificantibodies are then cloned by standard techniques of limiting dilutionand single cell manipulation and grown up to produce milligrammequantities of the secreted immunoglobulins. The secreted antibodies arethen characterised by ion exchange chromatography (Wong JT and Colvin RB(1987) J. Immunol. 139, 1369) and purified for experimental diagnosticuse. In the present example, affinity chromatography was used forimmunoglobulin purification.

Preparation of assay Bispecific immunoglobulin or enrichedimmunoglobulin secreted by fusomas is immobilized on multiwell plates byincubation in ELISA coating buffer. Plates are blocked with 10% (V/V)FCS in PBS. The antibody is then loaded with enzyme by incubation withenzyme containing media. Unbound enzyme is removed by washing and theantibody enzyme complex is then ready for use.

The complex is used in two different ways to measure antigen. Firstantigen is added for 15 minutes, the supernatant removed and thissupernatant then assayed for the presence of enzyme activity releasedfrom the complex. Secondly, in simultaneous one step format, the enzymesubstrate is added to the complex at the same time as the antigen. Inboth cases, enzyme activity is measured directly by the colour changeassociated with substrate conversion is indicative of the presence ofantigen in the sample.

For example, lung surfactant apoprotein A purified by density-dependentcentrifugation (Katyal SL and Singh G (1979) Lab Invest 40 562) is usedto calibrate the assay. Samples of amniotic fluid from prematuredeliveries are then assayed for apoprotein concentration.

Bispecific antibody demonstrating antibody-mediated signal transduction

Fusoma cell line GAL 30.19 secretes a bispecific immunoglobulin reactivewith SP-A and β-galactosidase (from Escherichia coli). The cell line wasisolated from a cell fusion event between 6-thioguanine resistant D4hybridoma (Randle et al 1992 in preparation), subclone D4tg13 secretingan antibody reactive with SP-A and splenocytes from a BALB/c femalemouse immunized weekly over an eight week period with 10 μg per dose ofbeta-galactosidase (Sigma G5635) supported with an alum adjuvant. 20 μgof beta-galactosidase was given intravenous four days prior to the cellfusion experiment.

Cell fusion was performed by standard techniques and resulting cellmixture was plated in HAT selection medium. Cultures were screened 17days later. 714 fusoma cultures were obtained, 41 of which were found tosecrete antibody reactive with betagalactosidase by indirect ELISA. 8cultures secreted immunoglobulin reactive with both SP-A andbeta-galactosidase as determined by Western Immunoblot. These cultureswere cloned by limited dilution and 6 clonal cultures were selected forfurther study. One of these cell lines GAL 30.19 is now described. Asample of GAL30.19 was deposited in accordance with the provisions ofthe Budapest Treaty at the European Collection of Animal Cell Cultures,Porton Down United Kingdom on 22nd April 1992 and has been accorded theaccession number 92042211.

The cell line was routinely grown in alpha HAT medium and producesapproximately 5 μg per ml of immunoglobulin in unstirred monolayerculture growth conditions. Enriched GAL 30.19 immunoglobulin wasisolated by standard affinity chromatography techniques using Protein ASepharose (Sigma P3391). Briefly, 1.2 litres of culture supernatant wasadjusted to pH8.2 by addition of 1M Tris HC1, pH8.5, and run on to a 6mlProtein A sepharose column. After washing with 10 volumes of PBS,adjusted to pH8.2 by addition of 1M Tris HC1, pH8.5, boundimmunoglobulin was eluted by use of sodium citrate buffer, pH3.5 0.1M.1ml fractions were immediately neutralized with 700 μl of 1M Tris HC1pH8.5. Protein concentrations of the eluted fractions were determined byCoomassie Blue dye binding assay and antibody titre estimated byindirect ELISA. 6.05 mg of immunoglobulin was isolated from 1.2 litresof culture medium. Antibody titre of the most concentrated fraction was1:10⁶ for beta-galactosidase and 1:10⁵ for SP-A by indirect ELISA.

Antigen capture to demonstrate recognition of both beta-galactosidaseand SP-A

Enriched GAL30.19 can be used in an antigen capture ELISA format todetect beta galactosidase and SP-A. Briefly, immunoglobulin was coatedat 5 μg per ml in carbonatebicarbonate buffer, pH9.6, 50 μl per well, ina 96 well flat bottom immunoassay plate (Falcon Cat No. 3912) overnightincubation at 4° C. Plates were blocked with 100 μl per well, 10% (v/v)FCS in PBS, 2 hours at room temperature.

Beta Galactosidase Antigen Capture:

Increasing concentrations of beta galactosidase were loaded in 50 μlvolumes, from 0-100 μg per ml, and incubated for 1 hour at roomtemperature. Wells were washed twice with 200 μl PBS 0.5% (v/v) PBSTween 20 and then bound beta galactosidase was detected by addition ofenzyme substrate, "beta galactosidase substrate buffer". The substratecomprised 20.5 mg of Onitrophenyl β-D-galactopyranoside (Sigma N-1127;ONPG) dissolved in 1 ml of 0.1M pH 7.3 phosphate buffer with gentlewarming. 832 μl of ONPG solution was added to 5 ml of phosphate buffercontaining bsa and magnesium chloride in the ratio of:

2.7 ml 0.1M pH 7.3 phosphate buffer:

0.1 ml 0.03M magnesium chloride with 0.5% (w/v) bovine serum albumin(bsa).

In antigen capture format, GAL 30.19 detected a minimum of 5 μg per mlof beta galactosidase.

SP-A Antigen Capture:

Concentrations of SP-A, from 5 to 100 μg per ml, were loaded in 50 μlvolumes and incubated for 1 hour at room temperature. Wells were washedtwice with PBS Tween 20 and bound SP-A detected by addition of 50 μl perwell 1:30 E8 biotin in PBS. E8 hybridoma secretes a monoclonal antibodyreactive with a second, distinct from D4, epitope of SP-A (Randle et al1992 in preparation). E8 immunoglobulin was substituted in theapproximate ratio of 3 biotin molecules per immunoglobulin: StockE8-Biotin was 1 mg per ml for dilution). After incubation for 30minutes, plates were washed twice with PBS Tween and wells were thenincubated with 50 μl of 1:500 Avidinalkaline phosphatase in PBS (1 mgper ml stock in PBS: Sigma A2527) for 30 minutes at 4° C. Wells werewashed three times with PBS Tween and then presence of alkalinephosphatase was detected by substrate conversion of para-nitro phenylphosphate, disodiumhexahydrate (Sigma 104-105E). Briefly, 50 μl per wellof the substrate was added at 1 mg per ml in 1M diethanolamine bufferpH9.8, "alkaline phosphatase substrate". The alkaline phosphatasesubstrate buffer comprises diethanolamine buffer 10% (v/v), consistingof 97ml diethanolamine, 800ml water, 100mg of magnesium chloridehexahydrate. 1M hydrochloric acid is added until the pH is 9.8, volumeis then made up to 1 litre with water. Stored in dark at 4° C. untiluse. Substrate conversion by enzyme was detected by measurement ofoptical density at 410nm. Using this antigen capture format, GAL 30.19can detect a minimum of 6.25 μg per ml SP-A.

GAL30.19 blocks the activity of beta galactosidase 50 μl enrichedGAL30.19 immunoglobulin at 1 mg per ml was added to 50 μl of betagalactosidase solution in PBS, concentration of 500 μg per ml. 100 μl ofbeta galactosidase substrate was added and substrate conversion wasmonitored at 410 nm. A control experiment using 50 μl of PBS in place ofthe antibody solution was performed in parallel. After 5 minutes,optical density of the enzyme product was 0.920 in the absence ofantibody and 0.597 in the presence of GAL30.19 in the test sample. Thisdemonstrates that GAL30.19 blocks the enzymic activity of betagalactosidase.

Purification of bispecific GAL30.19 imunoglobulin to homogeneity

Homogeneous bispecific immunoglobulin was isolated from enrichedantibody by sequential affinity chromatography. The method of choice issequential Affinity Chromatography. The immunoglobulins carrying thefirst antigenic site were .isolated by affinity chromatography using abead matrix carrying purified SP-A. Elution was performed using standarddiethylamine pH11, 1M, buffer and fractions neutralized with Tris-HC1,pH8, 1M. The neutralized fractions were "desaired" by buffer exchange toPBS using G25 Sephadex (trade mark) filtration. The samples were thensubjected to a second affinity chromatography step, using achromatography gel where the gel matrix carries a bead matrix carryingβ-galuctosidase. DEA elution was performed and the homogenous bispecificantibody desalted and stored in PBS 0.02% azide at 4° C. until used. Oncompletion of the chromatography 2.7 mg of enriched immunoglobulinyielded 0.38 mg of homogeneous immunoglobulin. Antibody titre of themost concentrated fraction was 1:10⁴ for beta galactosidase and 1:10³for SP-A. Presence of heavy and light chain polypeptides of GAL 30.19was confirmed by electrophoresis of the homogeneous sample in 10% (w/v)SDS PAGE under reducing conditions.

Demonstration of Antibody-Mediated Signal Transduction

Transducing antibody activity has been demonstrated both with enrichedGAL 30.19 immunoglobulin, isolated by Protein A affinity chromatographyand with purified immunoglobulin, isolated to homogeneity from enrichedantibody by sequential affinity chromatography on SP-A Sepharose andbeta galactosidase Sepharose.

EXAMPLE 1. Enriched Immunoglobulin Assay. (See FIG. 3)

Enriched GAL30.19 immunoglobulin was coated at 50 μg per ml in ELISAcoating buffer, carbonate/bicarbonate pH9.6, 50 μl per well, overnightat 4° C. Plates were blocked with 100 μl per well of 10% (V/V) FCS inPBS, 2 hours at room temperature. Wells were then incubated with 50 μlof 20 μg per ml beta galactosidase (Sigma G5635) in wash buffer, PBSwith 0.5% (W/V) bsa (Sigma A7888) for 1 hour at room temperature. Wellswere then washed with 2 washes of 200 μl PBS Tween 20 (0.05% V/V), toremove unbound enzyme from the immobilized transducing antibody complex.

50 μl volumes of increasing concentrations of SP-A, the specificantigen, KLH, a non-specific antigen of 800 KD alton molecular weightand mouse immunoglobulin μ, IgM a non specific antigen of 1000 kDmolecular weight from 6.25 to 100 μg ml⁻¹, diluted in wash buffer, werethen loaded into duplicate wells. After 15 minutes, the supernatant wasremoved to assess release of enzyme from the complex by β-galactosidasesubstrate conversion. 50 μl volumes of the test were incubated with 50μl of β-Galactosidase substrate buffer. Conversion from substrate toproduct was measured by optical density at 410 nm, indicating thepresence of released enzyme in the supernatant.

Release of enzyme from the transduction complex was measured in thesupernatant by β-galactosidase substrate conversion and measurement ofproduct optical density at 410nm. Only in the presence of SP-A, whichhas a molecular weight of 1200kD altons, and not in the presence ofantigens of similar molecular weight KLH -800 kD and Ig M - 1000kDaltons was enzyme released. The effect is titratable and, at higherconcentrations of SP-A, a saturation effect is noted.

In this format, the GAL 30.19 transducing antibody detects a minimum of6.25 μg ml⁻ SP-A.

EXAMPLE 2. Purified Bispecific Immunoglobulin Assay

Demonstrating Enzyme Release in the Presence of Specific Antigen (SeeFIG. 4).

Purified GAL30.19 immunoglobulin was coated at 20 μg per ml in ELISAcoating buffer, carbonate/bicarbonate pH9.6, 50 μl per well, overnightat 4° C. Plates were blocked with 100 μl per well of 10% (v/v) FCS inPBS, 2 hours at room temperature. Wells were then incubated with 50 μlof 20 μg per ml of beta galactosidase (Sigma G5635) in wash buffer, PBSwith 0.5% (w/v) bovine serum albumen (Sigma A7888), for 1 hour at roomtemperature. Wells were then washed with 2 washes of PBS Tween, toremove unbound enzyme from the immobilized Transducing Antibody complex.

50 μl volumes of increasing concentrations of SP-A, the specificantigen, and KLH, the non-specific antigen of equivalent molecularweight, from 6.25-100 μg per ml diluted in wash buffer, were then loadedinto duplicate wells. After 15 minutes the supernatant was removed toassess release of enzyme from the complex by beta galactosidasesubstrate conversion.

Briefly, 50 μl volumes of the test were incubated with 50 μl ofβ-galactosidase substrate buffer. Conversion of substrate to product wasmeasured by optical density at 410nm, indicating the presence ofreleased enzyme in the supernatant. Significant release of betagalactosidase from the transducing complex only occurs in the presenceof the specific antigen SPA, and not in the presence of an antigen ofsimilar molecular weight, KLH.

EXAMPLE 3 Demonstrating Purified Bispecific Immunoglobulin AssayOne-Step Antibody-Mediated Signal Transduction (See FIG. 5).

Transducing antibody complex was prepared as above and unbound betagalactosidase washed from the plates by two washes of PBS Tween.Simultaneous enzyme release on specific antigen detection was thendemonstrated as follows:

50 μl volumes of increasing concentrations of SP-A, the specificantigen, and KLH, the non-specific antigen of equivalent molecularweight, were prepared from 6.25-100 μg per ml diluted in wash buffer andmixed with 50 μl volumes of beta galactosidase substrate buffer. The 100μl samples of mixed antigen and beta galactosidase substrate were thenadded to wells containing the immobilized transducing antibody complex.Enzyme activity, from release of beta galactosidase from the complex,was measured by optical density at 410nm, colour being produced byenzyme mediated product formation.

Product formation was measured immediately following addition of samples(0') and at ten minutes (10'). In both cases, only presence of thespecific antigen SP-A, recognized by GAL30.19, results in, significantproduct formation. This clearly indicates that for GAL 30.19 homogeneousimmunoglobulin, antigen detection results in enzyme release in a onestep manner, with signal transduction of inactive bound enzyme to activebeta galactosidase capable of substrate

I claim:
 1. An immunoassay method for determining the presence orabsence of a first antigen in a sample, the method comprising:contactingthe sample with at least one antibody arranged to provide a first and asecond antigen binding site, said first antigen binding site beingarranged to bind a first antigen and said second antigen binding sitebeing arranged to bind a second antigen wherein binding of said firstantigen to said first antigen binding site causes release of said secondantigen bound to said second antigen binding site: and measuring theamount of second antigen released.
 2. The method of claim 1 wherein thesecond antigen binding site is on the same antibody as said firstantigen binding site.
 3. The method of claim 2 wherein said firstantibody is bound to a surface using a solution of the antibody having aconcentration of protein of greater than about 20 μg/ml.
 4. The methodof claim 2 wherein said at least one antibody is selected from the groupconsisting of a bispecific, a trispecific, or other multispecificantibody.
 5. The method of claim 1 wherein said first and second antigenbinding sites are on different antibody molecules.
 6. The method ofclaim 5 wherein said first and second antibodies are bound to a surfaceusing a solution of the antibody having a concentration of protein ofgreater than about 20 μg/ml.
 7. The method of claim 1, 4, 5, wherein thesecond antigen is an enzyme or other detectable marker.
 8. The method ofclaim 3 or 6 wherein the protein concentration of the solution isbetween about 50 and 100 μg/ml.
 9. The method of claim 3,or 6 whereinthe surface is that of a well of a microtiter tray.
 10. The method ofclaim 1 wherein the second antigen is enzyme which is bound in anenzymatically inactive form to second antigen binding site or isinactivated by binding to said site, and wherein said second antigen isreleased from said site in an active form upon binding of the firstantigen to the first antigen binding site.
 11. The method of claim 1wherein release of the second antigen from the second antigen bindingsite is followed by binding of the released second antigen or a reactionproduct of the released second antigen to a third antigen binding site,wherein binding to said third antigen binding site causes release of adetectable bound third antigen from an adjacent fourth antigen bindingsite.
 12. The method of claim 1, wherein a third antigen, if present inthe sample, binds to a third antigen binding site causing release of afourth antigen, wherein said second and fourth antigens are enzymeswhich when released from their respective antigen binding sites togethercatalyze a reaction cascade which produces a detectable product.
 13. Animmunoassay method for determining the presence or absence of anantigert in a sample, the method comprising:contacting the sample with amultispecific antibody having binding sites for the antigen and anenzyme, wherein binding of the enzyme to the antibody inactivates theenzyme, and wherein binding of the antigen to the antibody results inrelease of said enzyme in an active form from the antibody; anddetecting the activity of the released enzyme which indicates thepresence of the antigen in the sample.
 14. The method of claim 13wherein the antibody is a bispecific antibody.
 15. The method of claim13 or 14 wherein the enzyme is bound to the antibody by means of saidenzyme's active site.
 16. The method of claim 13 or 14 wherein theantigen is lung surfactant apoprotein.
 17. The method of claim 13 or 14wherein the enzyme is selected from the group consisting of:β-galactosidase, glucose oxidase, urease, carbonic anhydrase, andhorseradish peroxidase.
 18. A method of detecting lung surfactantapoprotein A in a sample of mammalian body fluid, comprising:contactingthe sample with a multispecific antibody having binding sites for lungsurfactant apoprotein A and an enzyme, wherein binding of the enzyme tothe antibody inactivates the enzyme, and wherein binding of lungsurfactant apoprotein A to the antibody results in release of saidenzyme in an active form from the antibody; and detecting the activityof the released enzyme which indicates the presence of lung surfactantapoprotein A in the sample.
 19. The method of claim 18 wherein theantibody is bispecific.
 20. The method of claim 19 wherein the enzyme isβ-galactosidase.
 21. A kit for use in an immunoassay method, said kitcomprising:a first antibody having a first and second antigen bindingsite for a first antigen and a second antigen, respectively, whereinsaid second antigen is an enzyme and binding to said second antigenbinding site inactivates the enzymatic activity of the second antigen; asecond antibody having: (a) a third antigen binding site for the secondantigen or a reaction product of the second antigen, and (b) a fourthantigen binding site for a detectable third antigen; wherein binding ofthe first antigen to the first antibody causes release of the secondantigen in an enzymatically active form, the released second antigen ora reaction product thereof being capable of binding to said secondantibody, thereby causing release of said detectable third antigen fromsaid second antibody.
 22. The kit of claim 21 wherein the antibodies arebound to the surface of a biosensor.
 23. A trispecific antibody for usein an immunoassay method, comprising:a first antigen binding sitedirected to a diagnostic marker, a second antigen binding site directedto a first enzyme, and a third antigen binding site directed to a secondenzyme, wherein the first and second enzymes, when bound to the secondand third antigen binding sites, are in an enzymatically inactive form,wherein upon binding of the diagnostic marker to the first antigenbinding site the first and second enzymes are released from the secondand third antigen binding sites in enzymatically active form, wherein areaction product of the released second enzyme is a substrate for thereleased first enzyme, and said first enzyme produces a detectablereaction product.
 24. The antibody of claim 23 which is bound to thesurface of a biosensor.
 25. A prebound complex consisting of amultispecific antibody and an enzyme, said multispecific antibodycomprising binding sites for an antigen and an enzyme, wherein theenzyme is bound to and inactivated by binding to the antibody andwherein said enzyme can be released from the antibody in an active formupon binding of the antigen to the antibody.
 26. The brebound complex ofclaim 25 wherein said antibody is bispecific.
 27. A prebound complexconsisting of a multispecific antibody and an enzyme, said multispecificantibody comprising binding sites for lung surfactant apoprotein A andan enzyme, wherein the enzyme is bound to and inactivated by binding tothe antibody and wherein said enzyme can be released from the antibodyin an active form upon binding of lung surfactant apoprotein A to theantibody.
 28. The prebound complex 27 of claim 27 wherein said antibodyis produced by the cell line GAL30.19 as deposited in accordance withthe provisions of the Budapest Treaty at the European Collection ofAnimal Cell Cultures, Porton Down, United Kingdom, under the accessionnumber
 9204221. 29. The prebound complex of claims 25 or 27 wherein themultispecific antibody is bound to the surface of a biosensor.
 30. Thecell line GAL30.19 as deposited in accordance with the provisions of theBudapest Treaty at the European Collection of Animal Cell Cultures,Porton Down, United Kingdom, under the accession number 92042211.